1. Field of the Invention
This invention refers to a process for obtaining epilutein and, more specifically to a process for obtaining 3xe2x80x2-epilutein from lutein-containing extracts, and to a process for the production of optically active zeaxanthin from said 3xe2x80x2-epilutein.
2. Description of the Related Art
More than 50 years have elapsed since Karrer and Jucker published an article in Helv. Chim. Acta 30, 266 (1949) with respect to the first isomerization of an epsilon-endgroup in a carotenoid molecule into one with a beta-endgroup with the aid of a strong alkali. This includes an extension of the length of the polyene-chain from originally 10 to 11 conjugated double bonds, as illustrated in FIG. 1 of the enclosed drawings.
However, only a few of the scientific laboratories in the world realized the importance of this invention, and so, the reaction generally fell into oblivion, and was regarded merely as a curiosity. In recent times the situation has changed drastically, mainly because of the following reasons:
1. The recognition of the general importance of the carotenoids as colorants for animal tissues either by ingestion from natural sources or from food additives [J. C. Bauernfeind, G. B. Brubacher, H. M. Klxc3xa4ui, W. L. Marusich, xe2x80x9cUse of Carotenoidsxe2x80x9d, in xe2x80x9cCarotenoidsxe2x80x9d(Ed. O. Isler, H. Gutmann, U. Solms), Birkhxc3xa4user, Basel, 1971, Pag. 743,ff; J. C. Bauernfeind (ed), xe2x80x9cCarotenoids as Colorants and Vitamin A Precursorsxe2x80x9d, Academic Press, N.Y. 1981; K. Schiedt, xe2x80x9cAbsorption and Metabolism in Birds, Fish and Crustaceansxe2x80x9d, in xe2x80x9cCarotenoidsxe2x80x9d, Ed. G. Britton, S. Liaaen-Jensen, H. Pfander, Vol. 3: Biosynthesis and Metabolismxe2x80x9d Birkhxc3xa4user, Basel, 1998, pag. 285, ff].
2. Significant progress in the experimental and practical handling of the epsilon-beta-arrangement (U.S. Pat. No. 5,523,494 issued Jun. 4, 1996 to Torres-Cardona et al., see also chronological survey of relevant references in this reference).
The situation today shows a fairly good and industrially useful conversion of lutein (I) (see FIG. 1) into a stereoisomer of zeaxanthin which itself proves to be a better colorant than lutein (I), e.g. for broilers and shrimps. This is very important because lutein is found in abundant quantities in yellow flowers and in all green leaves.
However, a main disadvantage of this rearrangement exists in the stereochemical situation: lutein from plant sources always has the (3R,3xe2x80x2R.6xe2x80x2R)-chirality [R. Buchecker, C. H. Eugster, Chimia, 25, 192 (1971)] as shown in formula I at FIG. 1. Therefore, zeaxanthin prepared from lutein necessarily has the (3R,3xe2x80x2S)-chirality as depicted in II (see FIG. 2) and, consequently, is the meso-form [R. Buchecker, P. Hamm, C. H. Eugster, Chimia, 26, 134 (1972); A. G. Andrewes, G. Borch, S. Liaaen-Jensen, Acta Chem. Scand. B28,139 (1974)]. A trifling quantity of (3R,3xe2x80x2R)-zeaxanthin found in zeaxanthin prepared from lutein is derived from optically active zeaxanthin which naturally accompanies lutein in Tagetes, [U.S. Pat. No. 5,780,693 issued Jul. 14, 1998 to Bernhard K. et al.] and is not a product of an epimerization.
The main disadvantage of meso-zeaxanthin is caused by its lower potency in pigmenting of egg yolk, as shown by the following references: K. Schiedt, xe2x80x9cAbsorption and Metabolism in Birds, Fish and Crustaceansxe2x80x9d, in Carotenoidsxe2x80x9d, (Ed. G. Britton, S. Liaaen-Jensen, H. Pfander), Vol. 3: Biosynthesis and Metabolismxe2x80x9d, Birkhxc3xa4user, Basel, 1998, pag. 285 ff., and H. Hencken, Poultry Science, 71, 711-717, (1992):
On the other hand, Garnett et al, U.S. Pat. No. 5,747,544 issued May 5, 1998, discloses the convenience of obtaining (3R-3xe2x80x2R) stereoisomers of zeaxanthin, for treating or preventing retinal degeneration in humans, by administering a drug formulation containing said (3R-3xe2x80x2R) stereoisomers of zeaxanthin in a carrier substance.
From these results, there followed the necessity of developing a procedure to obtain optically-active III from lutein (I).
The conversion of meso-zeaxanthin into III (see FIG. 3), or the racemate or the (3S,3xe2x80x2S)-zeaxanthin, to applicants knowledge, has no precedent, and no such publication has been found in the literature. It would require a selective protection of one of the two OH-groups, e.g. by acetylation followed by an enzymic hydrolysis. This, hopefully, could lead to 25% of the desired product at best.
Otherwise, instead of enzymic reaction, an inversion of the stereochemistry at the unprotected OH-group could be envisaged, e.g. by an Mitsunobu-reaction. However, the necessary reagents are costly and further, the yields in this reaction are usually low; see hereafter.
Accordingly, Applicants could not see the purpose of expending time and effort in the experimental testing of such a reaction.
Another remarkable way is described by Sanroma et al. (U.S. Pat. No. 5,998,678 issued Dec. 7, 1999 to Sanroma et al.) which mentions the oxidation of meso-zeaxanthin (II) into the dioxocompound IV (see FIG. 4) followed by a hydride reduction into the mixture of II and racemic zeaxanthin. Applicants do not see recommendable this multi-step sequence, mainly because they do not see a more efficient way to carry it out, as discussed below.
In further reflecting on these problems with racemic zeaxanthin, applicants focused on 3xe2x80x2-epilutein (V, see FIG. 5) as a possibly excellent starting material for the preparation of optically active (3R,3xe2x80x2R)-zeaxanthin (III), provided it also permitted one to carry on the epsilon-beta-rearrangement with alkali.
Overview of the Occurrence of 3xe2x80x2-epilutein in Nature and of the Preparation of 3xe2x80x2-epilutein
A search in libraries and in data banks proved that the occurrence of 3xe2x80x2-epilutein in plants is extremely rare.
Until the present time, it has only been detected in the following: flowers of Caltha palustris [A. G. Dabbagh, K. Egger, Zeitschr. Pflanzenphysiol. 72, 177 (1974)], anthers of roses and peonies [E. Marki-Fischer, C. H. Eugster, Helv. Chim. Acta 37, 1205 (1990)], and flowers of Tagetes [F. Khachik, A. Steck, H. Pfander; J. Agric. Food Chem. 47,455 (1999)].
It occurs partly in esterified form. Common by-products are carotenes and carotenoles. From this, it follows that plants do not offer a reasonable source for the preparative isolation of 3xe2x80x2-epilutein.
In animal tissues and liquors 3xe2x80x2-epilutein is more widespread, but unfortunately, always in very low concentration; see the overview provided in T. Matsuno, T. Maoka, M. Katsuyama, T. Hirono, Y. Ikuno, M. Shimizu, T. Komori, Comp. Biochem. Physiol. B85, 77 (1986). Recent findings with respect to 3xe2x80x2-epilutein concern:xe2x80x94human plasma [F. Khachik, G. R. Beecher, M. B. Goli, W. R. Lusby, J. C. Smith jr., Anal. Chem. 64, 2111 (1992)],xe2x80x94the skin of trouts [M. C. Vecchi, G. Englert, H. Mayer, Helv. Chim. Acta, 65, 1950 (1982)];xe2x80x94human breast milk [F. Khachik, C. J. Spangler, J. C. Smith jr., L. M. Canfield, A. Steck, H. Pfander, Anal. Chem. 69, 1873 (1997)].
The small quantities found made any preparative isolation prohibitively costly.
Synthesis of several epiluteins starting from lower synthons are described in H. Mayer xe2x80x9cCarotenoid Chemistry and Biochemistryxe2x80x9d Ed. G. Britton, T. W. Goodwin, Pergamon Press, London 1982, pag. 55, ff for various epimers, but not for 3xe2x80x2-epilutein itself.
A conversion of lutein into 3xe2x80x2-epilutein via 3xe2x80x2-O-didehydrolutein (oxolutein, VI) followed by a hydride reduction was described for the first time in R. Buchecker, C. H. Eugster, A. Weber, Helv. Chim. Acta 61, 1962 (1978). It leads to a mixture of I:V with a ratio of 1:2 (I:V). The separation of both stereoisomers is easily performed by HPLC. Pure V was isolated by column-chromatography and fully characterized by melting point and relevant spectra.
In Caltha palustris 3xe2x80x2-epilutein is accompanied by lutein and oxolutein (VI) (see FIG. 6) [R. Buchecker, C. H. Eugster, Helv. Chim. Acta 62, 2817 (1979)], and [E. Mxc3xa4rki-Fischer, C. H. Eugster, Helv. Chim. Acta 73, 1205 (1990)].
This fact points to a special enzymic oxido-reduction in the plant.
Epimerization Reactions at C-3xe2x80x2 in Lutein.
The so-called xe2x80x9cacid-labilityxe2x80x9d of lutein has long been recognized, see for example F. W. Quackenbush, H. Steebock, W. A. Peterson, J. Amer. Chem. Soc. 60, 2937 (1938); H. H. Strain, xe2x80x9cLeaf Xanthophyllsxe2x80x9d, Carnegie Inst. Of Plant Biology, Washington, 1938, pag. 87.; A. L. Curl, Food Research 21, 689 (1956), but any identification of the products formed was lacking. Only Zechmeister et al, were able to identify some of the products they had produced by some very special elimination reactions [L. Zechmeister, J. W. Sease, J. Amer. Chem. Soc. 65, 1951 (1943); F. J. Petracek, L. Zechmeister, J. Amer. Chem. Soc. 78, 1427 (1956); L. Zechmeister, xe2x80x9cFortschr. Chem. Org. Naturstoffexe2x80x9d, 15, 31(1958)].
From a modern point of view, specific reactions at C-3xe2x80x2-OH of lutein are due to the specific nature of the allylic alcohol. In mechanistic terms its reaction with various electrophiles has to be classified as an SN1-type. This includes a planar allylic cation (VII, see FIG. 7) as intermediate which is prone to an attack by nucleophiles either from xe2x80x9cabovexe2x80x9d or xe2x80x9cbelowxe2x80x9d and both at C(3xe2x80x2) or C(5xe2x80x2). However, in contrast to J. Szabolcs, Acta Chim. Hung. 61, 301 (1969), applicants never found C(5xe2x80x2)-substituted products in their experiments.
In all likelihood, all of these reactions are reversible.
From mechanistic considerations it is clear that the orientation of an attack of the nucleophile depends also on steric factors, therefore, applicants always have to expect more than one product; either one with a cis or a transrelation to the substituent at C(6xe2x80x2).
Whether the reaction is thermodynamically or kinetically controlled is as yet unclear.
At this point a remark has to be made about the so called Mitsunobu-reaction. It works in the sense of an SN2-reaction with the result of a clean epimerization at the center concerned. Such a reaction with the intention of preparing 3xe2x80x2-epilutein from (unprotected) lutein has been published by H. R. Sliwka, S. Liaaen-Jensen, Acta Chem. Scand. B41, 518 (1987). Besides many by-products, only 0.3% of 3xe2x80x2-epilutein could be isolated.
Finally performing acid-catalyzed reactions of lutein in the presence of the nucleophillic solvent methanol allowed the isolation of lutein-3xe2x80x2-methylether (VIII see FIG. 8) [S. Liaaen-Jensen, S. Hertzberg, Acta Chem. Scand. 20, 1703 (1966)], but neither its stereochemistry nor the structure of other possible isomers were clarified.
In fact, such a reaction is, as discussed above, not stereoselective.
Applicants own experiments showed the presence of both cis- and trans-methylethers in a ratio of 2:1 [C. H. Eugster, Report by E-Mail on Nov. 11, 1999 to Industrial Orgxc3xa1nica, S.A. de C.V., Monterrey, Mxc3xa9xico]. They can be easily separated on a HPLC column.
Based on these facts, applicants were astounded to realize that until now, no single researcher has made use of the very common nucleophile OHxe2x88x92 in an SN1-reaction with lutein. From this fact it follows that a solution of lutein in a solvent that is also miscible with water, a reaction at C(3xe2x80x2)+ with water should occur during the addition of an aqueous solution of a strong acid with the formation of two stereoisomers, namely lutein (I) and 3xe2x80x2-epilutein (V). An excellent solvent for such a reaction is tetrahydrofurane in which lutein is easily soluble. Another very important fact is to avoid mineral acids whose anions show appreciable nucleophilic activity. Therefore, aqueous sulfuric acid or perchloric acid, etc., were Applicants first choice.
With a concentrate from Tagetes, containing 39% of lutein, Applicants were able to obtain a mixture of lutein and 3xe2x80x2-epilutein in a ratio of about 1:4 to about 1:5.5.
Other combinations could include e.g. glycolethers, dichloromethane, benzene, with aqueous sulfuric acid, perchloric acid, trifluoroacetic acid, ion-exchanger, etc.
The Epsilon-beta Rearrangement with 3xe2x80x2-epilutein (V) into (3R,3xe2x80x2R)-zeaxanthin (III)
In the case of 3xe2x80x2-epilutein possibly no assistance of C(3 xe2x80x2)xe2x80x94Oxe2x88x92 to the abstraction of Hxe2x80x94C(6xe2x80x2) takes place for stereochemical reasons, so a change of the conditions disclosed in U.S. Pat. No. 5,523,494, issued Jun. 4, 1996 to Torres-Cardona et al. was necessary.
In fact, under modified conditions, Applicants were able to obtain (3R,3xe2x80x2R)-zeaxanthin (III) in good yield and with a high optical purity from the mixture of 3xe2x80x2-epilutein with lutein. The residual lutein led to minor contamination of the product with meso-zeaxanthin (II).
However, until the present time, the proportion of (3R-3xe2x80x2R) stereoisomers of zeaxanthin obtained from the conventional saponification processes, is at most 3-7% of the total xanthophylls.
Applicants have had the foresight to realize the convenience of employing an optically active zeaxanthin for enhancing the color pigmentation of the broiler skin and egg yolks, as well as its use in the treatment or prevention of macula degeneration in humans.
It is therefore a main object of the present invention, to provide a process for obtaining 3xe2x80x2-epilutein, from a lutein-containing extract.
It is also a main object of the present invention, to provide a process for obtaining 3xe2x80x2-epilutein, of the above-disclosed nature, by reacting a lutein-containing extract, with an inorganic or organic acid, whose anions possess a very low nucleophilicity in order to obtain 3xe2x80x2-epilutein.
It is additionally an object of the present invention, to provide a process for obtaining 3xe2x80x2-epilutein, of the above-disclosed nature, from which optically active zeaxanthin is obtained.
It is a further main object of the present invention, to provide a process for obtaining optically active zeaxanthin, from 3xe2x80x2-epilutein.
It is still a main objective of the present invention, to provide a process for obtaining optically active zeaxanthin, by reacting 3xe2x80x2-epilutein with a strongly alkaline aqueous solution.